The kinetic properties and sensitivities to inhibitors of lactate dehydrogenases (LDH1 and LDH2) from Toxoplasma gondii: comparisons with pLDH from Plasmodium falciparum.

Toxoplasma gondii differentially expresses two forms of lactate dehydrogenase in tachyzoites and bradyzoites, respectively, designated LDH1 and LDH2. Previously it was demonstrated that LDH1 and LDH2 share a unique structural feature with LDH from the malarial parasite Plasmodium falciparum (pLDH), namely, the addition of a five-amino acid insert into the substrate specificity loops. pLDH exhibits a number of kinetic properties that previously were thought to be unique to pLDH. In the present study, kinetic properties of LDH1 and LDH2 were compared with those of pLDH. LDH1 and LDH2 exhibit broader substrate specificity than pLDH. For both LDH1 and LDH2, 3-phenylpyruvate is an excellent substrate. For LDH2, 3-phenylpyruvate is a better substrate even than pyruvate. By comparison, pLDH does not utilize 3-phenylpyruvate. Both LDH1 and LDH2 can utilize the NAD analog 3-acetylpyridine adenine dinucleotide (APAD) efficiently, similar to pLDH. LDH1 and LDH2 are inhibited competitively by a range of compounds that also inhibit pLDH, including gossypol and derivatives, dihydroxynaphthoic acids, and N-substituted oxamic acids. The lack of substrate inhibition observed with pLDH is also observed with LDH2. By comparison, LDH1 differs from LDH2 in exhibiting substrate inhibition in spite of an identical residue (M163) at a cofactor binding site that is thought to be critical for production of substrate inhibition. For gossypol and gossylic iminolactone, but not the other gossypol derivatives tested, the in vitro inhibition of T. gondii LDH activity correlated with specific inhibition of T. gondii tachyzoite growth in fibroblast cultures.

Simple and efficient method for measuring anti-toxoplasma immunoglobulin antibodies in human sera using complement-mediated lysis of transgenic tachyzoites expressing beta-galactosidase.

A simple and efficient method using transgenic Toxoplasma gondii tachyzoites expressing beta-galactosidase was developed for detection of specific antibodies against the parasite in sera of patients. The titers obtained with the new test were similar to those obtained with the Sabin-Feldman dye test run in parallel. Although significant changes in endpoint titers were not observed when sera drawn sequentially at 2- to 3-week intervals were tested with both procedures, apparent differences in antibody affinity were observed with the new test which were not perceptible with the Sabin-Feldman dye test. Like the Sabin-Feldman dye test, the new test is based on complement lysis of tachyzoites, but it is much easier to perform and the reaction is read colorimetrically instead of visually.

Strain typing of Toxoplasma gondii: comparison of antigen-coding and housekeeping genes.

Molecular characterization of Toxoplasma gondii isolates is central for understanding differences in disease transmission and manifestations. Only 3 subgroups (lineages) have been discerned with subtle within-lineage variation, permitting low-resolution classification of isolates. Because proteins, coding sequences, and especially antigen-coding genes have been used extensively in previous studies, we focused on sequence variation in introns of housekeeping genes, which may be more informative for phylogenetic analysis because they evolve under lower selection. We compared sequence variation in introns of 5 housekeeping genes with 2 antigen-coding genes. Introns of housekeeping genes were slightly more polymorphic than coding and noncoding regions of antigen-coding genes and only the former showed intralineage variation. Intragenic linkage disequilibrium was complete, but intergenic linkage, although highly significant, was incomplete, suggesting that genes are partially uncoupled. Six of 7 substitutions found within the region coding for the tachyzoite surface antigen, SAG2, were nonsynonymous, indicating that diversifying selection acts on this locus. Typing isolates on the basis of housekeeping and antigen-coding genes was consistent, but the phylogenetic relationships among the resulting groups was inconsistent. A cougar isolate typed as lineage II using a restriction fragment length polymorphism assay possessed multiple unique polymorphisms, suggesting that it represents a new lineage. We concluded that introns of housekeeping genes are preferred markers for phylogenetic study, and that multilocus genotyping is preferred for typing parasites, especially from feral or unstudied environments.

Use of recombinant antigens for detection of Toxoplasma gondii infection in swine.

Five recombinant Toxoplasma gondii antigens, designated B427, C51, C55, V22, and MBP30 were assessed for their potential use in an enzyme-linked immunoassay (EIA) for detection of T. gondii infection in swine. The antigens were evaluated with sera from young pigs that had been fed 1-10,000 T. gondii oocysts of the VEG or GT-1 strains. Results were compared with an EIA using a native T. gondii antigen extract. All 5 recombinant antigens, as well as native antigen, detected antibody responses as soon as 3 wk after infection in pigs inoculated with 1 or 10 oocysts of the VEG strain. This antibody response persisted, at varying levels, for 14 wk when the experiment was terminated. All antigens also detected antibody responses in pigs 4 wk after inoculation with 10,000 oocysts of the GT-1 strain. The antibody response recognized by native antigen remained high through 51 wk after inoculation. However, there was considerable animal-to-animal variation in responses to the individual recombinant antigens. Only antigens C51 and MBP30 consistently detected a positive antibody response over the entire 51-wk course of the experiment. These results suggest that these antigens might be useful for the serological detection of T. gondii infection in pigs.

Toxoplasma gondii expresses two distinct lactate dehydrogenase homologous genes during its life cycle in intermediate hosts.

Two Toxoplasma gondii genes were characterized that are differentially expressed during the parasite's life cycle. The genes named LDH1 and LDH2, respectively, encode polypeptides similar to the enzyme lactate dehydrogenase (LDH; L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) from a variety of organisms. They show 64.0% nucleotide identity in the coding region and both have an intron at the same relative position. The deduced amino acid sequences of LDH1 and LDH2 share 71.1% identity. LDH1 and LDH2 are most similar to an LDH of Plasmodium falciparum (46.5% and 48.5% amino acid identities, respectively). The mRNA of LDH2 was only detected in the bradyzoite stage, while the mRNA of LDH1 was detected in both the bradyzoite and tachyzoite stages. However, by isoelectric focusing and immunoblot analysis, only one LDH isoform was found to be expressed in each stage. Furthermore, the expression of a reporter gene carrying chloramphenicol acetyltransferase (CAT) coding sequence and the putative LDH2 promoter sequence was significantly up-regulated by growing parasites in tissue culture in media with alkaline pH (pH 8.2, a condition known to induce the expression of bradyzoite-specific antigens), while the expression of a CAT reporter construct carrying the putative LDH1 promoter sequence was down-regulated by similar treatment. These results indicate that LDH expression is developmentally regulated in T. gondii and suggest a possible correlation between stage conversion and alteration in carbohydrate or energy metabolism in this parasite.

Use of a recombinant antigen, SAG2, expressed as a glutathione-S-transferase fusion protein to immunize mice against Toxoplasma gondii.

The capacity of Toxoplasma gondii surface protein SAG2 to induce protective immunity against the parasite in mice was studied using recombinant SAG2 expressed as a glutathione-S-transferase (GST) fusion protein incorporated into immune stimulating complexes (iscoms). Immunization with the iscoms resulted in the production of antibodies recognizing SAG2 as well as GST. After oral challenge infection with T. gondii oocysts or tissue cysts, no protective effect was observed. On the contrary, mice immunized with fusion SAG2 or with GST iscoms died earlier than non-immunized control mice.

An immunohistochemical method for detecting bradyzoite antigen (BAG5) in Toxoplasma gondii-infected tissues cross-reacts with a Neospora caninum bradyzoite antigen.

The previously cloned gene of a bradyzoite-specific antigen (BAG5) of Toxoplasma gondii was used to express a fusion protein for subsequent antiserum production in rabbits. The BAG5 antiserum was used in an immunohistochemical procedure to look for reactive epitopes in bradyzoites and tachyzoites of T. gondii within animal tissues. Encysted bradyzoites in brain were stained deeply and diffusely. Although most unencysted organisms in brain were not stained, occasional free organisms had mild to deep staining. There was no staining of tachyzoites in liver where cysts were not observed. Neospora caninum organisms within animal tissues were also examined using the BAG5 immunohistochemical procedure. The BAG5 antiserum cross-reacted with N. caninum bradyzoites but had no affinity for tachyzoites.

Regulated secretion of multi-lamellar vesicles leads to formation of a tubulo-vesicular network in host-cell vacuoles occupied by Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that actively invades virtually all types of nucleated cells, surviving within a specialized vacuole called the parasitophorous vacuole. Shortly after invasion, the parasite modifies this vacuole by secreting a variety of proteins from electron-dense storage granules. Additionally, the parasite forms a network of membranous tubules within the lumen of the vacuole and connecting with the vacuolar membrane. We have used immunolabeling and cell fractionation to examine the secretion of two dense granule proteins, GRA1 and GRA2, which are involved in formation of the intravacuolar network. Following host-cell invasion, GRA1 was secreted into the lumen of the vacuole as a soluble protein that subsequently became peripherally associated with the network. In addition to being secreted as a soluble protein from dense granules, GRA2 was secreted within multi-lamellar vesicles released from a specialized posterior invagination of the parasite. The multi-lamellar vesicles assemble to form the intravacuolar network, which contains an integral membrane form of GRA2. These findings indicate that Toxoplasma has a highly developed regulated exocytosis pathway that modifies the parasitophorous vacuole by secretion of soluble proteins and by a novel process of membrane secretion.

Molecular characterization of a 65-kilodalton Toxoplasma gondii antigen expressed abundantly in the matrix of tissue cysts.

We describe the cloning and characterization of a novel antigen expressed in the bradyzoite stage of Toxoplasma gondii. A cDNA library was constructed in bacteriophage lambda gt11 Sfi-Not using messenger RNA molecules isolated from cysts of the ME49 strain of T. gondii. The recombinant phage library was subjected to screening with polyclonal antibodies against bradyzoite antigens. This screening identified a recombinant antigen that was recognized strongly by polyclonal antibodies against bradyzoite antigens as well as by sera from mice chronically infected with T. gondii. The native antigen is a protein of 65 kDa that localized to the matrix of the cyst and the cyst wall surrounding the bradyzoites. The antigen was found to be expressed abundantly in cysts but could not be detected in tachyzoites or within the parasitophorous vacuole of tachyzoite infected host cells. Genomic and cDNA sequence of the gene revealed an open reading frame encoding 452 amino acids interrupted by 2 introns: a 503-bp intron located in the 5' untranslated region preceding the protein coding sequence and a 110-bp intron located 95 bp downstream of the first ATG.

Two alleles of the gene encoding surface antigen P22 in 25 strains of Toxoplasma gondii.

A group of 25 strains of Toxoplasma gondii (4 mouse-virulent strains and 21 mouse-avirulent strains) were tested by immunoblot with 4 monoclonal antibodies (MAb) against surface antigen P22. Parasite lysates from only 12 strains were recognized by all 4 MAbs, while lysates from the remaining 13 strains (all avirulent) were not recognized by any of the 4 MAbs. Strains not recognized by the 4 MAbs were found to express an altered form of the P22 antigen. Sequencing of the P22 genes from 10 strains revealed only 2 alleles. One allele is identical to the gene from the virulent RH strain. The second allele carries 5 single nucleotide substitutions and an insertion of a GGT triplet when compared to the allele from the RH strain. Four of the 5 nucleotide changes result in amino acid substitutions and the triplet insertion results in an extra glycine residue. Four of the single base changes also result in restriction fragment length polymorphisms (RFLP). RFLP analysis of the P22 gene revealed only 2 patterns among the 25 strains. The allele of the P22 gene correlated with reactivity of the 4 MAbs to lysates of each strain. However, the allele of the P22 gene did not correlate with the virulence of each strain for mice.

Detection of Toxoplasma gondii in cerebrospinal fluid from AIDS patients by polymerase chain reaction.

The polymerase chain reaction (PCR) was used to detect Toxoplasma gondii DNA in cerebrospinal fluid (CSF) from 14 patients with AIDS by amplification of the repetitive B1 gene. Positive PCRs were obtained in CSF from four of nine patients with toxoplasmic encephalitis. CSF samples from five control patients were negative for T. gondii DNA by PCR.

Expression, characterization, and serologic reactivity of recombinant surface antigen P22 of Toxoplasma gondii.

The utility of recombinant Toxoplasma gondii surface antigen P22 for the detection of specific T. gondii antibodies in human sera was evaluated. Polymerase chain reaction was used to produce a 438-bp fragment of the P22 gene; the fragment corresponded to the amino acids predicted to be in the processed, native antigen. The fragment was subcloned into pGEX-2T and was expressed in Escherichia coli as a glutathione-S-transferase (GST) fusion protein. The fusion protein was purified in a soluble form and was found to be recognized by sera from infected individuals in immunoblots and an enzyme-linked immunosorbent assay. Immunoglobulin G antibodies in sera from 31 acutely infected patients in general reacted more strongly to the fusion protein than did those in sera from 31 patients with the chronic infection. None of the sera from a panel of 26 seronegative controls reacted with the fusion protein was separated from the GST partner by cleavage with thrombin, it retained its immunoreactivity and its electrophoretic mobility in polyacrylamide gels was found to be similar to that of native P22. By a modification of the published method for purification of the foreign polypeptide from the GST carrier, the recombinant P22 was readily purified to homogeneity by thrombin cleavage of the fusion protein while it was adsorbed to glutathione agarose.

Cloning, expression, and cDNA sequence of surface antigen P22 from Toxoplasma gondii.

Immunoblot, immunofluorescence, and complement-mediated cytolytic assays revealed that two new monoclonal antibodies raised against a membrane-enriched fraction of Toxoplasma gondii tachyzoites recognize protein P22 on the surface of the parasite. Using these monoclonal antibodies to screen a cDNA expression library in lambda gt11, several clones expressing recombinant fusion proteins were isolated. Subsequent screening of the library with a synthetic oligonucleotide derived from the 5' end of one of these cDNAs permitted the isolation of additional nonexpressing clones containing the entire translated sequence. Blots of parasite RNA and DNA suggested that the corresponding gene occurs as a single copy in the tachyzoite genome. The amino acid sequence deduced from the composite cDNA indicates a primary translation product with a theoretical molecular weight of 18,959. As expected for surface protein P22, the putative polypeptide contains a predicted N-terminal signal sequence and a C-terminal hydrophobic region characteristic of proteins attached to the membrane by a glycophospholipid anchor. Recombinant fusion proteins produced by the expressing clones were recognized on immunoblots by IgG antibodies in the sera of humans with acute and chronic T. gondii infection. Antibodies selected by the fusion protein reacted predominantly with a 22-kDa antigen on immunoblots of parasite lysate.

Filamentous fusion phage cloning vectors for the study of epitopes and design of vaccines.

Foreign sequences can be inserted within the minor coat protein, pIII, of filamentous phage, creating a fusion protein that is incorporated into the virion; the virions, which we call "fusion phage," display the foreign amino acids encoded in the insert on their surface. Phage bearing specific antigenic determinants from a target gene can be purified in infectious form from a vast excess of phage bearing other determinants by affinity to antibody directed against the gene product. Fusion phage can be used as a source of antigen as a carrier-hapten conjugate for obtaining immunological reagents in rabbits, and for B epitope mapping. By generating a fusion phage library expressing virtually all possible short, amino acid sequences, it may be possible to study epitopes on immunologically important proteins without the use of synthetic peptides and without ever having cloned the genes.

Antibody-selectable filamentous fd phage vectors: affinity purification of target genes.

Foreign DNA fragments can be inserted into a minor coat protein gene of filamentous phage, creating a fusion protein that is incorporated into the virion; we call these particles "fusion phage". The foreign amino acids are displayed on the surface, allowing fusion phage bearing antigenic determinants from a target gene to be purified in infectious form by affinity to antibody directed against the gene product. Here we introduce fusion-phage vectors that accept foreign DNA inserts with little effect on phage function; and describe affinity purification of virions bearing a target determinant from a 10(8)-fold excess of phage not bearing the determinant, using minute amounts of antibody. These "antibody-selectable" vectors are a promising alternative to conventional expression systems for using antibodies to clone genes, though the ability to isolate rare clones from actual libraries remains to be demonstrated.